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Abstract:

The invention provides a cancer chemotherapeutic agent which has fewer
side effects and excellent efficacy. The cancer chemotherapeutic agent of
the invention includes a cholestanol derivative represented by formula
(1):
##STR00001##
(wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,
Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof, and an
anti-cancer agent.

2. The cancer chemotherapeutic agent according to claim 1, wherein, in
formula (1), G is GlcNAc-Gal-.

3. The cancer chemotherapeutic agent according to claim 1, wherein the
anti-cancer agent is one or more species selected from the group
consisting of a taxane anti-cancer agent, a platinum complex anti-cancer
agent, a pemetrexed compound, and fluorouracil.

4. The cancer chemotherapeutic agent according to claim 3, wherein the
anti-cancer agent is one or more species selected from the group
consisting of Paclitaxel, Docetaxcel, Pemetrexed, 5-FU, Cisplatin,
Oxaliplatin, Cyclophosphamide, and Irinotecan.

5. The cancer chemotherapeutic agent according to claim 1, which is a
compounding agent.

6. The cancer chemotherapeutic agent according to claim 1, which is in
the form of a kit including a drug containing a cholestanol derivative
and a drug containing an anti-cancer agent.

7. The cancer chemotherapeutic agent according to claim 6, wherein the
drug containing a cholestanol derivative is a liposomal formulation.

8. A method of producing a chemotherapeutic agent for the treatment of
cancer, by combining a cholestanol derivative represented by formula (1):
##STR00006## wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-,
Fuc-Gal-, Gal-Glc-, or Gal- or a cyclodextrin inclusion compound thereof
and an anti-cancer agent.

9. A method for cancer chemotherapy, comprising administering, in
combination, a cholestanol derivative represented by formula (1):
##STR00007## wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-,
Fuc-Gal-, Gal-Glc-, or Gal- or a cyclodextrin inclusion compound thereof
and an anti-cancer agent, to a patient in need thereof.

10. The method for cancer chemotherapy according to claim 9, wherein the
cholestanol derivative or a cyclodextrin inclusion compound thereof and
the anti-cancer agent are administered to a patient in need thereof
simultaneously, or separately and intermittently.

11. The method for cancer chemotherapy according to claim 9, wherein, in
formula (1), G is GlcNAc-Gal-.

12. The method for cancer chemotherapy according to claim 9, wherein the
anti-cancer agent is one or more species selected from the group
consisting of a taxane anti-cancer agent, a platinum complex anti-cancer
agent, a pemetrexed compound, and fluorouracil.

13. The method for cancer chemotherapy according to claim 12, wherein the
anti-cancer agent is one or more species selected from the group
consisting of Paclitaxel, Docetaxcel, Pemetrexed, 5-FU, Cisplatin,
Oxaliplatin, Cyclophosphamide, and Irinotecan.

14. The method for cancer chemotherapy according to claim 9, wherein the
drug containing a cholestanol derivative is a liposomal formulation.

15. The method for cancer chemotherapy according to claim 9, wherein the
anticancer agent is Cisplatin.

16. The cancer chemotherapeutic agent according to claim 1, wherein the
anticancer agent is Cisplatin.

Description:

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a chemotherapeutic agent for
cancer (hereinafter referred to as a "cancer chemotherapeutic agent")
and, more particularly, to a cancer chemotherapeutic agent employing a
cholestanol derivative and an anti-cancer agent in combination.

[0003] 2. Background Art

[0004] A variety of anti-cancer agents used in chemotherapy for cancer,
which is one mode of cancer therapy, have hitherto been developed and
classified based on structure, action mechanism, etc. However, the
efficacy of such an anti-cancer agent employed as a single agent is
unsatisfactory. Instead, multi-drug therapy employing a plurality of
anti-cancer agents has been predominantly carried out in recent years
from the viewpoint of suppressing adverse side effects, and the effecacy
of multi-drug therapy has been recognized.

[0005] Under such circumstances, both of the development of new
anti-cancer combination chemotherapy, which has fewer adverse side effect
and higher efficacy than conventional chemotherapies, and the development
of new chemotherapeutic agents for use in the chemotherapy are desired.

[0006] Meanwhile, a cholestanol derivative, in which a sugar chain such as
GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-, Gal-, or GlcNAc- is
bonding to cholestanol (the compound that the double bond in the B ring
of the cholesterol is saturated), were previously found to have excellent
anti-tumor activity. JP-A-2000-191685, JP-A-1999-60592, WO 2005/007172
(pamphlet), and WO 2007/026869 (pamphlet) disclose the effects of such
cholestanol derivatives.

[0007] However, no cases have been reported in which the aforementioned
cholestanol derivatives and another anti-cancer agent are employed in
combination.

SUMMARY OF THE INVENTION

[0008] Thus, the present invention is directed to provision of a cancer
chemotherapeutic agent which has fewer side effects and excellent
efficacy.

[0009] In view of the foregoing, the present inventors have carried out
extensive studies, and have found that a remarkably potentiated
anti-cancer effect can be attained through employment, in combination, of
a cholestanol derivative represented by formula (1) or a cyclodextrin
inclusion compound thereof and a known chemotherapeutic agent
(anti-cancer agent), and thus the combined use of these pharmaceutical
agents in cancer chemotherapy is very useful.

[0010] Accordingly, the present invention is directed to the following (1)
to (10).

(wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,
Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof, and an
anti-cancer agent. (2) A cancer chemotherapeutic agent according to (1)
above, wherein, in formula (1), G is GlcNAc-Gal- or GlcNAc-. (3) A cancer
chemotherapeutic agent according to (1) or (2) above, wherein the
anti-cancer agent is one or more species selected from the group
consisting of a taxane anti-cancer agent, a platinum complex anti-cancer
agent, a pemetrexed compound, and fluorouracil. (4) A cancer
chemotherapeutic agent according to (3) above, wherein the anti-cancer
agent is one or more species selected from the group consisting of
Paclitaxel, Docetaxcel, Pemetrexed, 5-FU, Cisplatin, Oxaliplatin,
Cyclophosphamide, and Irinotecan. (5) A cancer chemotherapeutic agent
according to any of (1) to (4) above, which is a compounding agent. (6) A
cancer chemotherapeutic agent according to any of (1) to (4) above, which
is in the form of a kit including a drug containing a cholestanol
derivative and a drug containing an anti-cancer agent. (7) A cancer
chemotherapeutic agent according to (6) above, wherein the drug
containing a cholestanol derivative is a liposomal formulation. (8) Use,
in combination, of a cholestanol derivative represented by formula (1):

(wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,
Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof and an
anti-cancer agent, to a patient in need thereof. (10) A cancer
chemotherapy according to (9) above, wherein the cholestanol derivative
or a cyclodextrin inclusion compound thereof and the anti-cancer agent
are administered to a patient in need thereof simultaneously, or
separately at intervals.

[0011] Through employment of the cancer chemotherapeutic agent and the
cancer chemotherapy according to the present invention, prevention and
treatment of cancer can be realized with safety and higher efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a graph showing the cell proliferation inhibitory effects
of CDDP, GC-CD, and CDDP+GC-CD on colon 26 cells;

[0015] FIG. 4 is a graph showing the anti-tumor effect of single
administration of CDDP, GC-CD, and CDDP+GC-CD against peritoneal
dissemination caused by colon26 cells intraperitoneally inoculated in
mice;

[0016] FIG. 5 is a graph showing the anti-tumor effect of multiple
administration of CDDP, GC-CD, and CDDP+GC-CD against peritoneal
dissemination caused by colon26 cells intraperitoneally inoculated in
mice;

[0017] FIG. 6 is a graph showing the anti-tumor effect of single, delayed
administration of CDDP, GC-CD, and CDDP+GC-CD against peritoneal
dissemination caused by colon26 cells intraperitoneally inoculated in
mice, after confirmation of peritoneal dissemination on the mesothelium
of mice;

[0018]FIG. 7 is a graph showing the survival rate of mice to which
colon26 cells were intraperitoneally inoculated, upon single
administration of CDDP, GC-CD, or CDDP+GC-CD (single administration of
CDDP and double administration of GC-CD, respectively);

[0019] FIG. 8 is a graph showing the effect of suppressing or reducing the
tumor growth to which colon26 cells were subcutaneously inoculated in
mice, upon single administration of CDDP, GGC-CD, or CDDP+GGC-CD;

[0020] FIG. 9 is a graph showing the effect of inhibiting metastatis of
colon26 cells to the lung, upon single administration of CDDP, GC-CD,
GGC-CD, CDDP+GC-CD, and CDDP+GGC-CD;

[0021] FIG. 10-A is a graph showing the cell proliferation inhibitory
effects of an anti-cancer agent (5-FU, PTX, DTX, or CPT), GC-CD, and the
anti-cancer agent+GC-CD on colon 26 cells; and

[0022] FIG. 10-B is a graph showing the cell proliferation inhibitory
effects of an anti-cancer agent (L-OHP or CPA), GC-CD, and the
anti-cancer agent+GC-CD on colon 26 cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0023] The specific cholestanol derivatives represented by formula (1) and
employed in the present invention are all known compounds.

[0024] Among the cholestanol derivatives which are represented by formula
(1) and in which G is GlcNAc-Gal-, G is preferably
GlcNAcβ1,3-Galβ- or GlcNAcβ1,4-Galβ-. Among the
cholestanol derivatives (1) in which G is GlcNAc-Gal-Glc-, G is
preferably GlcNAcβ1,3-Galβ1,4-Glc-. Among the cholestanol
derivatives (1) in which G is Fuc-Gal-, G is preferably
Fucα1,3Gal-. Among the cholestanol derivatives (1) in which G is
Gal-Glc-, G is preferably Galβ1,4Glcβ-. Among the cholestanol
derivatives (1) in which G is Gal-, G is preferably Galβ-. Among the
cholestanol derivatives (1) in which G is GlcNAc-, G is preferably
GlcNAcβ--.

[0025] Of these, species in which G is GlcNAc-Gal- and GlcNAc- are more
preferred, with those in which G is GlcNAcβ1,4-Galβ- and
GlcNAcβ- being still more preferred.

[0026] The aforementioned cholestanol derivatives may be produced through
a method, for example, disclosed in the aforementioned Patent Documents
or a similar method.

[0027] The cholestanol derivative represented by (1) readily forms an
inclusion complex with a cyclodextrin or a derivative thereof. Thus, the
cholestanol derivative employed in the present invention may be a
cyclodextrin inclusion compound thereof. In formation of such inclusion
compounds, the size of the guest molecule to be included, Van der Waals
interaction between the guest molecule and cyclodextrin, and hydrogen
bond between the hydroxyl groups of cyclodextrin and the guest molecule
must be taken into consideration. Therefore, insoluble guest compounds do
not always form the corresponding inclusion compounds. However, the
cholestanol derivative of the present invention can form good inclusion
complexes with cyclodextrin.

[0028] Examples of the cyclodextrin forming the cyclodextrin inclusion
compound of the present invention include cyclodextrins such as
α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin; and
cyclodextrion derivatives such as methyl-β-cyclodextrin,
2-hydroxypropyl-β-cyclodextrin, monoacetyl-β-cyclodextrin, and
2-hydroxypropyl-γ-cyclodextrin. Of these,
2-hydroxypropyl-β-cyclodextrin is preferred for obtaining improved
solubility.

[0029] The cyclodextrin inclusion compound may be prepared through, for
example, the following procedure: an aqueous solution of a cyclodextrin
or a derivative thereof having an appropriate concentration (e.g., 20 to
40%) is prepared, and the cholestanol derivative of the present invention
is added to the aqueous solution, followed by stirring of the resultant
mixture.

[0030] No particular limitation is imposed on the concentration of the
solution of the cholestanol derivative (1), so long as the cholestanol
derivative can form an inclusion compound with cyclodextrin. Generally,
the concentration is about 1 to about 50 mass %, preferably about 10 to
about 30 mass %.

[0031] The thus-produced cyclodextrin inclusion compound is highly
water-soluble and, therefore, effectively exhibits the effect of the
guest in vivo. Another advantage of the cyclodextrin inclusion compound
is to ensure consistent in vitro test results.

[0032] Alternatively, the cholestanol derivative (1) may be prepared into
a liposomal formulation, whereby the cholestanol derivative can be more
effectively delivered to the action expression site. Another advantage of
the cyclodextrin inclusion compound is to ensure consistent in vitro test
results.

[0033] Preferably, the liposomal formulation includes the cholestanol
derivative of the present invention, a membrane component, and an
aliphatic or aromatic amine.

[0034] The cholestanol derivative content in the liposomal formulation is
preferably 0.3 to 2.0 mol, more preferably 0.8 to 1.5 mol, with respect
to 1 mol of the membrane component.

[0035] The membrane component may be a phospholipid. Specific examples of
preferably employed phospholipids include natural and synthetic
phospholipids such as phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, and phosphatidic acid; mixtures
thereof; and modified natural phospholipids such as aqueous lecithin.
Examples of more preferred species include phosphatidylcholine
(1α-dipalmitoylphosphatidylcholine (DPPC)).

[0036] The aliphatic or aromatic amine is employed mainly for positively
charging the surface of lipid membrane. Examples of such amines include
aliphatic amines such as stearylamine and oleylamine; and aromatic amines
such as fluorenethylamine. Among them, stearlylamine is particularly
preferably employed.

[0037] Preferably, the amine is contained in an amount of 0.04 to 0.15
mol, more preferably 0.1 to 0.15 mol, with respect to 1 mol of membrane
component (phospholipid).

[0038] In addition to the aforementioned components, if required, the
liposome may further contain a membrane structure stabilizer such as
cholesterol, fatty acid, diacetyl phosphate, etc.

[0039] The aqueous solution employing for dispersing the membrane
component is preferably water, physiological saline, buffer, aqueous
sugar solution, or a mixture thereof. Either an organic or an inorganic
buffer may be used, so long as the buffer has a buffering action in the
vicinity of the hydrogen ion concentration of body fluid. Examples of
such buffers include a phosphate buffer.

[0040] No particular limitation is imposed on the method of preparing the
liposomal formulation, and generally employed methods may be selected.
Examples of the employable method include methods disclosed in
JP-A-1982-82310, JP-A-1985-12127, JP-A-1985-58915, JP-A-1989-117824,
JP-A-1989-167218, JP-A-1992-29925, and JP-A-1997-87168; a method
disclosed in Methods of Biochemical Analysis (1988) 33, p. 337; or a
method disclosed in "Liposome" (published by Nankodo).

[0041] No particular limitation is imposed on the anti-cancer agent which
is used in combination with the cholestanol derivative represented by
formula (1) or a cyclodextrin inclusion compound thereof, and known
cancer chemotherapeutic agents may be used. Standard therapeutic agents
which have been established with respect to the cancer of therapy target
are preferably employed.

[0043] As shown in the Examples described hereinbelow, when the
cholestanol derivative represented by formula (1) or a cyclodextrin
inclusion compound thereof is used in combination with an anti-cancer
agent, proliferation of cancer cells of various types are strongly
suppressed, as compared with the case of administration of only each
agent. Therefore, this combined chemotherapy can drastically enhance
therapeutic efficacy and mitigation of adverse side effects, and a
pharmaceutical product containing these ingredients is a useful cancer
chemotherapeutic agent.

[0044] No particular limitation is imposed on the cancer which can be
effectively treated by administering the cancer chemotherapeutic agent of
the present invention. Examples of the target cancer include malignant
tumors such as gastric cancer, large bowel cancer, pancreatic cancer,
uterus cancer, ovaria cancer, lung cancer, gallbladder cancer, esophageal
cancer, liver cancer, breast cancer, mesothelioma, and prostatic cancer.

[0045] The form of the cancer chemotherapeutic agent of the present
invention may be a compounding agent in which the aforementioned
ingredients are mixed at an appropriate ratio, each at an effective
amount, to form a single dosage form (single-formulation type), or may be
a kit that consists of the respective dosage form of the aforementioned
ingredients, each of which is formed independently including each
effective amount, and that enables the dosage forms to be administered
simultaneously or separately at intervals (double-formulation type).

[0046] Similar to general pharmaceutical formulations, no particular
limitation is imposed on the dosage form of the above-described
formulation, and the form may be any of the solid form such as tablet,
liquid form such as injection, dry powder dissolving before use, etc.

[0047] No particular limitation is imposed on the administration route of
the formulation, and an appropriate route may be determined depending on
the dosage form of the agents. For example, an injection solution may be
administered intravenously, intramuscularly, subcutaneously,
intradermally, or interperitoneally, and a solid form may be administered
orally or enterally.

[0048] The formulation may be prepared through a known method in the art.
All pharmaceutically acceptable carriers (excipients or diluents such as
a filler, a bulking agent, and a binder) generally employed in the art
may also be employed.

[0050] An oral liquid formulation may be prepared by mixing the drug
ingredients of the present invention with a flavoring agent, buffer,
stabilizer, deodorant, etc., and forming the mixture into internal liquid
agent, syrup, elixir, etc. through a method known in the art. The
flavoring agent employed in the preparation may be any of the
aforementioned members. Examples of the buffer include sodium citrate.
Examples of the stabilizer include traganth, gum arabic, and gelatin.

[0051] Injection solutions may be prepared by mixing the drug ingredients
of the present invention with additives such as a pH-regulator, buffer,
stabilizer, tonicity agent, and local anesthetic agent, etc., and forming
the mixture through a method known in the art, to thereby provide
subcutaneous, intramuscular, and intravenous injection liquids. Examples
of the pH-regulator and buffer include sodium citrate, EDTA, thioglycolic
acid, and thiolactic acid. Examples of the local anesthetic include
procaine hydrochloride and lidocaine hydrochloride. Examples of the
tonicity agent include sodium chloride and glucose.

[0052] Suppositories may be prepared by mixing the drug ingredients of the
present invention with a carrier for formulation known in the art such as
polyethylene glycol, lanolin, cacao butter, and fatty acid triglyceride,
and with an optional surfactant such as Tween (registered trademark), and
forming the mixture into suppositories through a method known in the art.

[0053] Ointments may be prepared by mixing the drug ingredients of the
present invention with optional additives generally employed in the art
such as a base, stabilizer, moisturizer, and preservatives, and forming
the mixture into ointments through a method known in the art. Examples of
the base include liquid paraffin, white petrolatum, white beeswax,
octyldodecyl alcohol, and paraffin. Examples of the preservative include
methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, and propyl
p-hydroxybenzoate.

[0054] Cataplasms may be prepared by applying the aforementioned ointment,
gel, cream, paste, etc., to a generally employed support through a
routine method. Examples of appropriate supports include woven and
nonwoven fabric made of cotton, staple fiber, or chemical fiber, and film
and foamed sheet made of soft vinyl chloride, polyethylene, polyurethane,
etc.

[0055] Generally, the formulation is preferably prepared so as to have a
cholestanol derivative content and an anti-cancer agent content of 0.0001
to 80 wt. % (as effective ingredient).

[0056] When the cancer chemotherapeutic agent of the present invention is
provided as a kit, the kit may be designed to pack independently the
respective dosage form including separately the cholestanol derivative
represented by formula (1) or a cyclodextrin inclusion compound thereof
and an anti-cancer agent, each of which have been prepared in the above
manner, and to be used each pharmaceutical formulation taken separately
from the corresponding respective package before use. Alternatively, each
pharmaceutical formulation may be held in a package suitable for each
time of combined administration.

[0057] The dose of the cancer chemotherapeutic agent of the present
invention varies depending on the body weight, age, sex, symptoms of a
patient in need thereof, route and frequency of administration to a
patient in need thereof, etc. Generally, for example, the daily dose for
an adult is about 0.1 to 30 mg/kg as the cholestanol derivative (1),
preferably 3 to 10 mg/kg. The dose of the anti-cancer agent may fall
within a range established with respect to the agent, or may be lower
than that range.

[0058] No particular limitation is imposed on the frequency of
administration, and the agent may be administered once or several times a
day. Single administration a day is preferred. When the kit is used, each
of the formulation including separated drug ingredients may be
administered simultaneously or intermittently.

EXAMPLES

[0059] The present invention will next be described in more detail by way
of examples, which should not be construed as limiting the invention
thereto.

Example 1

Effect of Drug Addition on Inhibition of Cancer Cell Proliferation

[0060] Colon26 cells (derived from mouse colon cancer) were inoculated to
a 96-well plate (1×104 cells/50 μL, 10% FCS-RPMI
medium/well), and incubated at 37° C. for 16 hours. To each well,
cisplatin (abbreviated as "CDDP") and/or a cyclodextrin inclusion
compound (abbreviated as "GC-CD") of a cholestanol derivative in which G
in formula (1) is GlcNAcβ-(abbreviated as "GC") was added
(multi-fold dilution by FCS(-)-medium: final concentration: ≦0.500
μM, 50 μL), followed by incubation at 37° C. for two days.
GC-CD was prepared in accordance with a method disclosed in Example 1(2)
in WO 2007/026869. Specifically, a 40% aqueous solution of
hydroxypropyl-β-cyclodextrin was prepared, and GC was added to the
solution, followed by mixing with stirring (80° C. for 30
minutes), to thereby prepare GC-CD.

[0061] As a control, wells to which only FCS(-)-medium had been added were
employed. Viable count was performed by means of a cell counting kit
(product of Dojin).

[0063] The procedure of Example 1 was repeated, except that colon26 cells
were changed to MKN45 (derived from human gastric cancer), NCIH226
(derived from human lung cancer), and Colo201 (derived from human colon
cancer). CPI rate (%) was determined in a similar manner. FIG. 2 shows
the results.

[0064] In Example 2, a cyclodextrin inclusion compound (abbreviated as
"GGC-CD") of a cholestanol derivative in which G in formula (1) is
GlcNAcβ1,4-Galβ- (abbreviated as "GGC") was also used. GGC-CD
was produced in a manner similar to the method as the aforementioned
GC-CD production method, except that the cholestanol compound was changed
to GGC. CPI rate with respect to the cancer cells was determined. FIG. 3
shows the results.

Example 3

Effect of Drug Addition on Inhibition of Cancer Cell Proliferation In Vivo

[0065] In the following Examples, Balb/c mice (6-weeks old, female) were
employed as test animals.

[0066] (1) Colon26 cells (1×104 cells/mouse) were
intraperitoneally inoculated to the mice (day 0). On the following day
after inoculation (day 1), CDDP and/or GC-CD was adjusted with
physiological saline (Otsuka normal saline) to a concentration of
interest, and CDDP, GC-CD, or CDDP+GC-CD (500 μL) was
intraperitoneally administered to the mice, followed by breeding. On day
19, mice were dissected, and the weight of the mesentery and the greater
omentum was measured. To the control group, only physiological saline
(500 μL) was administered (n=10; 10 mice/group).

[0067] FIG. 4 shows the results.

[0068] (2) Colon26 cells (1×104 cells/mouse) were
intraperitoneally inoculated to the mice (day 0). On day 1, day 2, day 3,
day 6, day 7, and day 8, CDDP and/or GC-CD was adjusted with
physiological saline (Otsuka normal saline) to a concentration of
interest, and CDDP, GC-CD, or CDDP+GC-CD (500 μL) was
intraperitoneally administered to the mice, followed by breeding. On day
21, mice were dissected, and the weight of the mesentery and the greater
omentum was measured. To the control group, only physiological saline
(500 μL) was administered (n=10; 10 mice/group).

[0069] FIG. 5 shows the results.

[0070] (3) Colon26 cells (1×104 cells/mouse) were
intraperitoneally inoculated to the mice (day 0). On day 7, CDDP and/or
GC-CD was adjusted with physiological saline (Otsuka normal saline) to a
concentration of interest, and CDDP, GC-CD, or CDDP+GC-CD (500 μL) was
intraperitoneally administered to the mice, followed by breeding. On day
18, mice were dissected, and the weight of the mesentery and the greater
omentum was measured. To the control group, only physiological saline
(500 μL) was administered (n=10; 10 mice/group).

[0071] FIG. 6 shows the results.

Example 4

Anti-Tumor Effect by Drug Addition

[0072] Balb/c mice (6 weeks old, female) were employed as test animals.
Colon26 cells (1×104 cells/mouse) were intraperitoneally
inoculated to the mice (day 0). On day 2 and/or day 3, CDDP and/or GC-CD
was adjusted with physiological saline (Otsuka normal saline) to a
concentration of interest, and CDDP (once, on day 2), GC-CD (twice, on
day 2 and 3), or CDDP (once, on day 2)+GC-CD (twice, day 2 and 3) (500
μL) was intraperitoneally administered to the mice, followed by
breeding. The survival duration (days) was counted to day 43. To the
control group, only physiological saline (500 μL) was administered
(n=10; 10 mice/group).

[0074] Balb/c mice (6 weeks old, female) were employed as test animals.
Colon26 cells (5×104 cells/mouse) were subcutaneously
inoculated to the mice (day 0). After confirmation that the tumor size
reached about 4 mm (day 7 to 10 after inoculation), CDDP and/or GGC-CD
was adjusted with physiological saline (Otsuka normal saline) to a
concentration of interest, and CDDP, GGC-CD, or CDDP+GGC-CD (200 μL)
was administered to the mice through the tail vein, followed by breeding.
Time-dependent change in tumor size was monitored to day 21, and the
corresponding tumor volume was determined. To the control group, only
physiological saline (200 μL) was administered (n=7; 7 mice/group).

[0075] FIG. 8 shows the results.

Example 6

Cancer Metastasis Inhibitory Effect by Drug Addition

[0076] Balb/c mice (6 weeks old, female) were employed as test animals.
Colon26 cells (5×104 cells/mouse) were intraperitoneally
inoculated to the mice (day 0). Immediately after inoculation, CDDP
and/or GC-CD or GGC-CD was adjusted with physiological saline (Otsuka
normal saline) to a concentration of interest, and CDDP, GC-CD (or
GGC-CD), or CDDP+GC-CD (or GGC-CD) (200 μL) was administered to the
mice through the caudal vein, followed by breeding. On day 14, mice were
dissected, and the tumor nodes in the lungs were counted. To the control
group, no substance was administered (n=10; 10 mice/group).

[0077] FIG. 9 shows the results.

Example 7

Effect of Drug Addition on Inhibition of Cancer Cell Proliferation

[0078] Colon26 cells (derived from mouse colon cancer) were inoculated to
a 96-well plate (1×104 cells/50 μL, 10% FCS-RPMI
medium/well), and incubated at 37° C. for 16 hours. To each well,
well-known anti-cancers agent (Oxaliplatin (abbreviated as "L-OHP"),
Fluorouracil (5-FU), Paclitaxel (TXL; abbreviated as "PTX"), Docetaxel
(TXT; abbreviated as "DTX"), Irinotecan (CPT-11; abbreviated as "CPT"),
or Cyclophosphamide (abbreviated as "CPA") and/or a cyclodextrin
inclusion compound (abbreviated as "GC-CD") of a cholestanol derivative
in which G in formula (1) is GlcNAcβ- (abbreviated as "GC") was
added (multi-fold dilution by FCS(-)-medium: final concentration:
≦500 μM, 50 μL), followed by incubation at 37° C. for
two days. GC-CD was prepared in accordance with a method disclosed in
Example 1(2) in WO 2007/026869. Specifically, a 40% aqueous solution of
hydroxypropyl-β-cyclodextrin was prepared, and GC was added to the
solution, followed by mixing with stirring (80° C. for 30
minutes), to thereby prepare GC-CD.

[0079] As a control, wells to which only FCS(-)-medium had been added were
employed. Viable count was performed by means of a cell counting kit
(product of Dojin).

[0081] As described hereinabove, through employment, in combination, of
the cholestanol derivative of the present invention or a cyclodextrin
inclusion compound thereof and an anti-cancer agent, proliferation of
various cancer cells is strongly inhibited, and synergistic effect and/or
effect of potentiating anti-tumor action of a known anti-cancer agent can
be obtained.

Patent applications by Shin Yazawa, Osaka-Shi JP

Patent applications by Takayuki Asao, Maebashi-Shi JP

Patent applications by Toyo Nishimura, Osaka-Shi JP

Patent applications by NATIONAL UNIVERSITY CORPORATION GUNMA UNIVERSITY